Substances

ABSTRACT

The present invention provides modified CD8 molecules whose binding to MHC is enhanced compared to wild type CD8, wherein Ser 53  of at least one CD8α chain thereof is mutated to another amino acid. It also provides nucleic acids encoding such molecules, and to the use of such molecules and nucleic acids in immunosuppressive therapy, in particular as inhibitors of cytotoxic T cell responses.

[0001] The present invention relates to modified CD8 proteins andnucleic acids encoding such proteins, and to their use inimmunosuppressive therapy, in particular as inhibitors of cytotoxic Tcell responses.

[0002] Major histocompatibility complex Class I and II proteins (MHC, orHLA in man) bind peptide antigens and present them on the cell surface.MHC Class I molecules are expressed to varying degrees on most nucleatedcells, while Class II expression is restricted to a subset of cellsreferred to as specialised antigen presenting cells (APCs). Class Imolecules present peptides derived from proteins expressed within thecell. Their role is to provide “markers” on the surface of cells toallow the immune system to monitor the state of these cells forabnormalities. Class II molecules obtain peptides derived from proteinstaken up by the APCs from the extracellular space. Their role can beconsidered to be to monitor the extracellular body fluids for foreignantigens. APCs include the interdigitating dendritic cells found in theT cell areas of the lymph nodes and spleen; Langerhan's cells in theskin; follicular dendritic cells in B cell areas of the lymphoid tissue;monocytes, macrophages and other cells of the monocyte/macrophagelineage; B cells and T cells; and a variety of other cells such asendothelial cells and fibroblasts which are not classical APCs but canact in the manner of an APC.

[0003] MHC-peptide complexes are recognised by T lymphocytes expressinga unique T cell receptor (TCR) matching the specific MHC-peptidecombination. T cell precursors enter the thymus where they undergo aselection procedure ensuring that T cells which respond to self-peptidesare eradicated (negative selection). In addition, T cells that do nothave the ability to recognise the MHC variants presented, fail to mature(positive selection).

[0004] Recognition of specific MHC-peptide complexes by T cells ismediated by the T cell receptor (TCR), which consists of an α and a βchain, both of which are anchored in the membrane. In a recombinationprocess similar to that observed for antibody genes, the TCR α and βgenes rearrange from Variable, Joining, Diversity and Constant elements,creating enormous diversity in the extracellular antigen binding domains(10¹³ to 10¹⁵ different possibilities). Antibody receptors and TCRs arethe only types of molecules that recognise antigens in a specificmanner. The TCR is the only receptor specific for particular peptideantigens presented in MHC, where the peptide is often the only sign ofan abnormality within a cell.

[0005] CD8 and CD4 are transmembrane glycoproteins characteristic ofdistinct populations of T lymphocytes whose antigen responses arerestricted by class I and class II MHC molecules, respectively. Theyplay major roles both in the differentiation and selection of T cellsduring thymic development and in the activation of mature T lymphocytesin response to antigen presenting cells. CD8 and CD4 are thereforeconsidered to be the main accessory molecules for T cell receptors andhence are referred to as co-receptors. Mature T cells expressing CD4 arereferred to as helper T cells while CD8⁺ T cells are called killer Tcells or cytotoxic T lymphocytes (CTLs). In addition to its role in thepositive selection of T cells during differentiation in the thymus, CD8is essential for the ability of most mature CTLs to kill target cells.

[0006] Both CD8 and CD4 are immunoglobulin superfamily proteins. Theydetermine antigen restriction by binding to MHC molecules at aninterface distinct from the region presenting the antigenic peptide, butthe structural basis for their similar functions appears to be verydifferent. Their sequence similarity is low and, whereas CD4 isexpressed on the cell surface as a monomer, CD8 is expressed as an ααhomodimer or an αβ heterodimer. CD8 contacts an acidic loop in the α3domain of Class I MHC, thereby increasing the avidity of the T cell forits target. CD8 is also involved in the phosphorylation events leadingto CTL activation through the association of its α chain cytoplasmictail with the tyrosine kinase p56^(lck).

[0007] Suppressors of the cellular arm of the immune system, such assuppressors of CD4 or CD8 T cells, are urgently needed for the treatmentof auto-immune disorders, such as rheumatoid arthritis, lupuserthymatosus, psoriasis vulgaris, ankylosing spondylitis, Reiter'sdisease, post-salmonella arthritis, post-shigella arthritis,post-yersinia arthritis, post-gonococcal arthritis, uveitis, amylodosis,idiopathic hemachromatosis and myasthenia gravis, as well as ofhypersensitivity (such as allergic reactions) and the prevention ofgraft rejection and graft-versus-host disease.

[0008] The therapeutic action of antibodies directed against CD4 and CD8has been assessed (De Fazio, et al. Transplantation 61: 104-10 (1996)),but with limited success and antibodies in general are not well suitedas drugs since they tend to induce secondary immune responses and areshort-lived. Administration of steroids is another way of suppressingthe immune system but their effect is extremely indirect and associatedwith severe side-effects.

[0009] It is known that soluble CD8 and derivatives thereof can causeinhibition of CTL. For example, Choksi, et al. (Nature Medicine 4:309-314 (1998)) used free CD8-derived peptides to inhibit CTL. Onepeptide in particular, “CSSHNKPC”, could inhibit both thedifferentiation and effector stages of CTL response. However, a veryhigh concentration of peptide (>100 μg/ml) was required to bring aboutthis inhibition (>50%). Soluble CD8 has also been shown to causeinhibition of CTL (W0 99/21576; Sewell, et al. Nature Medicine 5:399-404 (1999)). The inhibitory effect of the soluble CD8 molecule wasmore dramatic than that observed with an anti-CD8 monoclonal antibody.

[0010] Although these soluble CD8 molecules can prevent or inhibit CD8⁺T cell responses, they have a relatively low affinity for MHC/peptidecomplex. It is desirable to provide modified CD8-derived peptides andpolypeptides which have a higher affinity for MHC so as to enhance theCTL inhibitory effect of soluble CD8.

[0011] According to a first aspect of the present invention, there isprovided a modified CD8 molecule whose binding to MHC is enhancedcompared to wild type CD8, wherein Ser₅₃ of at least one CD8α chainthereof is mutated to another amino acid.

[0012] According to a second aspect, the present invention provides anucleic acid, particularly a DNA, comprising a sequence which encodes amodified CD8 molecule whose binding to MHC is enhanced compared to wildtype CD8, wherein Ser₅₃ is of at least one CD8α chain thereof is mutatedto another amino acid.

[0013] In a third aspect, the invention provides a compositioncomprising a modified CD8 molecule of the first aspect or a nucleic acidof the second aspect together with a pharmaceutically acceptablediluent, excipient or carrier.

[0014] In a fourth aspect, the invention provides a modified CD8molecule of the first aspect, or a nucleic acid of the second aspect,for use in medicine.

[0015] In a fifth aspect, the invention provides the use of a modifiedCD8 molecule of the first aspect, or of a nucleic acid of the secondaspect, in the manufacture of a medicament for modulating CD8⁺ T cellresponse.

[0016] In a sixth aspect, the invention provides a method of modulatingthe activation of a CD8⁺ T cell by a class I Major HistocompatibilityComplex (MHC), the method comprising exposing the class I MHC to amodified CD8 molecule of the first aspect. Nucleic acids of the presentinvention may be used to transfect cells to produce modified CD8molecules of the invention in vivo to modulate the activation of a CD8+Tcell.

[0017] In a seventh aspect, the invention provides a method for thetreatment of an autoimmune disorder, hypersensitivity (e.g. allergicreaction), graft versus host disease or graft rejection, comprisingadministering to a patient a modified CD8 molecule of the first aspector a nucleic acid of the second aspect.

[0018] In an eighth aspect, the present invention provides a productcontaining a modified CD8 molecule of the first aspect or a nucleic acidof the second aspect and an immunosuppressive agent as a combinedpreparation for simultaneous, sequential or separate use in modulatingCD8⁺ T cell response.

[0019] The present invention is described in more detail herein withreference to the accompanying drawings, in which:

[0020]FIG. 1a shows the amino acid and nucleic acid sequence of thewild-type human CD8αchain, FIGS. 1b and 1 c show the amino acid andnucleic acid sequences of respective soluble and membrane-bound CD8αmolecules of the present invention, and FIG. 1d shows the amino acid andnucleic acid sequences of a soluble form of the human CD8β monomer.

[0021]FIGS. 2a and c are graphs illustrating the affinity of wild typehuman CD8αα for Tax/HLA-A2/β2m complex, and FIGS. 2b and d are graphsillustrating the affinity of modified human CD8αα in which Ser₅₃ ismodified to Asn for Tax/LA-A2/β2m complex;

[0022]FIG. 3a is a graph illustrating the affinity of wild type humanCD8αα for Flu/HLA-A2/β2m complex, and FIG. 3b is a graph illustratingthe affinity of modified human CD8αα in which Ser₅₃ is modified to Asnfor Flu/HLA-A2/β2m complex; and

[0023]FIG. 4a is a graph illustrating the affinity of modified humanCD8αα in which Gln₂ is modified to Lys for Tax/HLA-A2/β2m complex, andFIG. 4b is a graph illustrating the affinity of modified human CD8αα inwhich Leu₉₇ is modified to Tyr for Tax/HLA-A2/β2m complex.

[0024] In the present invention, reference to numbered amino acidresidues in human CD8 is in accordance with the numbering of the aminoacid residues in FIG. 1a.

[0025] The modified CD8 molecules of the present invention provide animprovement over those disclosed previously due to their greater abilityto occupy the MHC binding site and inhibit CD8⁺ T cell response. In W099/21576, we suggested that knowledge of the molecular structure ofCD8αα and a MHC molecule (HLA-A2) meant that it would be possible todesign CD8 mutants which have increased binding to MHC, and suggestedcertain mutations which may have this effect. Surprisingly, we have nowfound that none of the suggested mutants has increased binding. Indeed,unexpectedly, we have found that of all of the potential mutationsavailable, only mutations of a single amino acid residue have thiseffect.

[0026] In one embodiment, Ser₅₃ is mutated to Asn. However, it will beappreciated that Ser₅₃ may be mutated to other amino acids, particularlythose with similar steric/electrostatic/polar properties to Asn. Forexample, Ser₅₃ may be mutated to Gln or His to create an additionalhydrogen bond between CD8 and HLA in the same way as Asn. Alternatively,Ser₅₃ may be mutated to Lys. This residue has an electrostaticattraction to Asp₂₂₇ of HLA.

[0027] It is preferred if the modified CD8 of the present invention isderived from human CD8. The sequences for two human forms of the CD8receptor, αα and αβ, are known (EMBL/GENBANK database accession numbers:CD8α, M27161; CD8β, X13444). The two forms of the receptor arefunctionally equivalent and no significant differences in the effects ofusing one or the other for immune inhibition would be expected.

[0028] The human CD8 gene expresses a protein of 235 amino acids. Theprotein can be considered to be divided into the following domains(starting at the amino terminal and ending at the carboxy terminal ofthe polypeptide):

[0029] signal peptide (amino acids −21 to −1)—this is cleaved off inhuman cells during the transport of the receptor to the cell surface andthus does not constitute part of the mature, active receptor;

[0030] immunoglobulin (Ig)-like domain (approximately amino acids1-115)—this domain assumes a structure, referred to as theimmunoglobulin fold, which is similar to those of many other moleculesinvolved in regulating the immune system, the immunoglobulin family ofproteins. The crystal structure of the CD8αα receptor in complex withthe human MHC molecule HLA-A2 has demonstrated how the Ig domain ofCD8αα receptor binds the ligand;

[0031] membrane proximal stalk region (amino acids 116-160)—this domainis thought to be an extended linker region allowing the CD8αα receptorto “reach” from the surface of the T-cell over the top of the MHC to thea3 domain of the MHC where it binds. The stalk region is glycosylatedand thought to be inflexible;

[0032] transmembrane domain (amino acids 161-188)—this anchors the CD8ααreceptor in the cell membrane and is therefore not part of the solublerecombinant protein;

[0033] cytoplasmic domain (amino acids 189-214)—this mediates asignalling function in T-cells through its association with p56^(lck)which is involved in the T cell activation cascade of phosphorylationevents.

[0034] In the present invention, the CD8 molecule generally has asufficient portion of the immunoglobulin domain to be able to bind toMHC. Generally, the molecule will comprise all or a substantial part ofthe native CD8α immunoglobulin domain, but may comprise at least 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110 or 115 amino acids of the immunoglobulin domain. The moleculesof the present invention are preferably dimers (i.e. αα or αβ), althoughCD8α monomer are included within the scope of the present invention. Inαα dimer molecules of the present invention it is preferred if both αchains carry the Ser₅₃ mutation.

[0035] The CD8 molecule may be a soluble form of the native CD8molecule. The term “soluble form” is used herein in relation to the CD8molecule in the manner in which it is conventionally used in the art inrelation to cell surface receptors. A soluble form of a cell surfacereceptor is usually derived from the native form by deletion of thetransmembrane domain. The protein may be truncated by removing both thecytoplasmic and the transmembrane domains, or there may be deletion ofjust the transmembrane domain with part or all of the cytoplasmic domainbeing retained. The protein may be modified to achieve the desiredsoluble form by proteolytic cleavage, or by expressing a geneticallyengineered truncated or partially deleted form. In one embodiment, theCD8 molecule of the present invention is a monomer or a homodimer of apolypeptide which comprises residues 1-120, except of course that Ser₅₃is mutated as described above. Alternatively, it may be a heterodimer ofsuch a polypeptide and a soluble form of the CD8β chain, for example asshown in FIG. 1d. In a further embodiment, Cys₃₃ of at least one α chainthereof is mutated to Ser or Ala. This mutation prevents the formationof inappropriate inter- or intra-chain disulphide bonds between Cys₃₃and the other Cys residues (Cys₂₂ and Cys₉₄). As a result, this mutanthas increased yield on expression and/or refolding. One CD8 molecule ofthe present invention is a monomer or a homodimer of a polypeptide whichhas the amino acid sequence as shown in FIG. 1b. Another is aheterodimer of such a polypeptide and a polypeptide having the aminoacid sequence of FIG. 1d.

[0036] It is however not essential that the CD8 molecule of the presentinvention is soluble, especially when the molecule is intended to beadministered using gene therapy. Such molecules may comprise all or partof the signal peptide and/or cytoplasmic and/or transmembrane domains.In one embodiment, the CD8 molecule of the present invention comprisesresidues 1-214 as shown in FIG. 1c. In another embodiment, the CD8molecule lacks the transmembrane domain (residues 161-188) and/orincludes the signal peptide. Homodimers of such molecules andheterodimers with corresponding β chains are also included within thescope of the invention.

[0037] Also included within the scope of the invention are the CD8molecules described above with one or more of the following variations.

[0038] Variations of the C-terminal truncation point. Longer or shorterversions of the receptor may be stable and functional. For solubleforms, there is no general rule to predict where the optimal truncationpoint for a soluble version of a transmembrane protein is. In the caseof CD8, the polypeptide could be between 1 and 15 amino acids longer orshorter. However, when shortened at the C-terminus, the molecule stillretains a short fragment of the membrane-proximal stalk region. Thesoluble CD8 could even comprise the cytoplasmic domain, having just thetransmembrane domain deleted. It is also envisaged that the C-terminuscould be fused to peptides or protein domains, such asglutathione-S-transferase, for purification purposes, or to a label fordetection, as is well known in the art. In the case of the protein ofFIG. 1b, 1-15 amino acid residues may be absent from the C-terminus, butwith at least a part of the membrane-proximal stalk region, i.e. theregion defined by amino acids 116-120, retained; all or part of thesequence “ala-pro-arg-pro-pro-thr-pro-ala” may be added at theC-terminus; and/or all or part of the CD8 cytoplasmic membrane peptidesequence may be added at the C-terminus.

[0039] Variations in the N-terminal truncation point. The N-terminaltruncation point could be varied, just like the C-terminal truncationpoint, without any influence on the functional effect of the protein. Itis also envisaged that the N-terminus could be fused to peptides orprotein domains, such as glutathione-S-transferase, for purificationpurposes, or to a label for detection, as is well known in the art. Inthe case of the protein of FIG. 1b, methionine maybe present at theN-terminus; 1-15 amino acid residues may be absent from the N-terminus;and/or all or part of the sequence “leu-leu-leu-his-ala-ala-arg-pro” maybe added to the N-terminus.

[0040] Conservative amino acid substitutions. A large number ofconservative amino acid substitutions can be introduced in the proteinwithout causing any significant changes. Thus, it may be possible toreplace one amino acid with another of similar “type”, for instance,replacing one hydrophobic amino acid with another. In the case of suchhomologues and derivatives, the degree of identity with a modified CD8as described above is less important than that the homologue orderivative should retain a mutation of Ser₅₃ and an enhanced binding toMHC is enhanced compared to wild type CD8. However, suitably, homologuesor derivatives having at least 60% identity are provided. Preferably,homologues or derivatives having at least 70% identity, more preferablyat least 80% identity are provided. Most preferably, homologues orderivatives having at least 90% or even 95% identity are provided.

[0041] The percent identity of two amino acid sequences or of twonucleic acid sequences is determined by aligning the sequences foroptimal comparison purposes (e.g. gaps can be introduced in the firstsequence for best alignment with the sequence) and comparing the aminoacid residues or nucleotides at corresponding positions. The “bestalignment” is an alignment of two sequences which results in the highestpercent identity. The percent identity is determined by the number ofidentical amino acid residues or nucleotides in the sequences beingcompared (i.e., % identity=number of identical positions/total number ofpositions×100).

[0042] The determination of percent identity between two sequences canbe accomplished using a mathematical algorithm known to those of skillin the art. An example of a mathematical algorithm for comparing twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programsof Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incorporatedsuch an algorithm. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilised as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can beused to perform an iterated search which detects distant relationshipsbetween molecules (Id.). When utilising BLAST, Gapped BLAST, andPSI-Blast programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.Another example of a mathematical algorithm utilised for the comparisonof sequences is the algorithm of Myers and Miller, CABIOS (1989). TheALIGN program (version 2.0) which is part of the CGC sequence alignmentsoftware package has incorporated such an algorithm. Other algorithmsfor sequence analysis known in the art include ADVANCE and ADAM asdescribed in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5;and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci.85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search.

[0043] More particularly, the invention provides a soluble CD8αα or ββmolecule containing a substantial part of the extracellular region ofCD8, including the immunoglobulin domain and a fragment of the membraneproximal stalk region, which CD8 molecule is not disulphide-linkedbetween the two chains of the molecule, wherein Ser₅₃ of at least one αchain thereof is mutated as described above.

[0044] The CD8 molecules described herein may be in the form of amultimer, that is two or more CD8 monomer or dimer (αα or αβ) moleculeslinked (covalently or otherwise) together. The CD8 molecules may beassociated with one another via a linker molecule. Alternatively oradditionally, the CD8 molecules may be attached to larger entities suchas membrane structures or particles.

[0045] Suitable linker molecules include multivalent attachmentmolecules such as avidin, streptavidin and extravidin, each of which hasfour binding sites for biotin. Thus, biotinylated CD8 molecules can beformed into multimer complexes of CD8 having a plurality of CD8 bindingsites. The number of CD8 molecules in the resulting complex will dependupon the quantity of CD8 in relation to the quantity of linker moleculeused to make the complexes, and also on the presence or absence of anyother biotinylated molecules. Preferred complexes are trimeric ortetrameric CD8 complexes. One or both chains of the CD8 molecule arepreferably biotinylated, conveniently by means of a biotinylationsequence expressed as a tag on the α or β chain. A preferred multimer isa tetramer with three mutant CD8 molecules and a fourth molecule, whichmay be a co-stimulatory agent, such as CD28 or CTLA-4.

[0046] Suitable structures for attachment of soluble CD8, optionallyalready in multimeric form, include membrane structures such asliposomes and solid structures which are preferably particles such asbeads. Other structures known to those skilled in the art which may beexternally coated with CD8 molecules are also suitable.

[0047] A modified CD8 molecule of the present invention may be providedin substantially pure form. For example, it may be provided in a formwhich is substantially free of other proteins. The modified CD8molecules of the present invention can be provided alone, as a purifiedor isolated preparation. They may be provided as part of a mixture withone or more other molecules of the invention.

[0048] The following tests for enhanced MHC binding for modified CD8molecules of the present invention compared to the wild-type moleculecan be performed. The binding of soluble modified CD8 molecules to MHCcan be determined by following the method detailed in Example 14 herein.The binding of full length modified CD8 molecules to MHC canconveniently be determined using the cell adhesion method detailed inSalter et al., Nature 338 (6213) 345-347. Briefly, this method involvesexpression of the modified CD8 molecule in a monolayer of CHO cells andmonitoring the binding of APCs to the CD8 expressing CHO cells. For bothof these tests the MHC binding affinity determination should by theaverage of three experiments.

[0049] Gene cloning techniques maybe used to provide a modified CD8molecule of the invention. These techniques are disclosed, for example,in J. Sambrook et al, Molecular Cloning 2nd Edition, Cold Spring HarborLaboratory Press (1989). Thus, the present invention provides a nucleicacid, particularly a DNA, comprising a sequence which:

[0050] (i) encodes a modified CD8 molecule as defined herein;

[0051] (ii) is an RNA equivalent of the DNA of (i);

[0052] (iii) is complementary to the sequences of (i) or (ii); or

[0053] (iv) has substantial identity with the sequences of (i), (ii) or(iii).

[0054] In one embodiment of this aspect of the invention, the nucleicacid has the DNA sequence set out in FIG. 1b or 1 c with or without thesignal sequence herein. The term “RNA equivalent” when used aboveindicates that a given RNA molecule has a sequence which iscomplementary to that of a given DNA molecule (allowing for the factthat in RNA “U” replaces “T” in the genetic code).

[0055] The nucleic acid molecules of the invention may include aplurality of such sequences. The skilled person will appreciate that thepresent invention can include novel variants of those particular novelnucleic acid molecules which are exemplified herein. Such variants areencompassed by the present invention. For example, additions,substitutions and/or deletions are included. In addition, andparticularly when utilising microbial expression systems, one may wishto engineer the nucleic acid sequence by making use of known preferredcodon usage in the particular organism being used for expression. Thus,synthetic or non-naturally occurring variants are also included withinthe scope of the invention.

[0056] Preferably, sequences which have substantial identity have atleast 50% sequence identity, desirably at least 75% sequence identityand more desirably at least 90 or at least 95% sequence identity withsaid sequences. In some cases, the sequence identity may be 99% orabove. Desirably, the term “substantial identity” indicates that saidsequence has a greater degree of identity with any of the sequencesdescribed herein than with prior art nucleic acid sequences.Substantially identical sequences may hybridise with the sequencesdescribed above under moderate or highly stringent hybridisingconditions. For a high degree of selectivity, relatively stringentconditions are used to form the duplexes, such as low salt or hightemperature conditions. As used herein, “highly stringent conditions”means hybridisation to filter-bound DNA in 0.5 M NaHPO₄, 7% sodiumdodecyl sulphate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1%SDS at 68° C. (Ausubel F. M. et al., eds., 1989, Current Protocols inMolecular Biology, Vol. I, Green Publishing Associates, Inc., and JohnWiley & Sons, Inc., New York, at p. 2.10.3) For some applications, lessstringent conditions for duplex formation are required. As used herein“moderately stringent conditions” means washing in 0.2×SSC/0.1% SDS at42° C. (Ausubel et al., 1989, supra). Hybridisation conditions can alsobe rendered more stringent by the addition of increasing amounts offormamide, to destabilise the hybrid duplex. Thus, particularhybridisation conditions can be readily manipulated, and will generallybe chosen depending on the desired results.

[0057] The nucleic acid molecules of the invention may be in isolated orrecombinant form. They may be incorporated into a vector and the vectormay be incorporated into a host cell. Such vectors and suitable hostsform yet further aspects of the present invention.

[0058] The invention provides, in a still further aspect, a method ofproducing a modified CD8 molecule of the invention, which methodcomprises the steps of: i) effecting expression of a nucleic acidmolecule of the present invention in a bacterium or eukaryotic cell andrecovering the expressed protein from a cell culture; and ii) treatingthe expressed protein to facilitate its purification and carrying outsaid purification. Preferably, the nucleic acid is modified via silentmutations designed to increase expression via the prevention of theformation of a 5′ hairpin secondary structure in the expressed mRNA.Preferably at step iii), the treatment of the expressed protein involvessolubilising the protein and treating the protein so as to cause it tofold into a form resembling its native state, which is then purified.Where the CD8 molecule is an αβ heterodimer, the nucleic acid sequenceof FIG. 1d may be expressed in addition to the mutant α chain.

[0059] A means of producing soluble CD8αα and CD8αβ in CHO cells for usein ligand binding studies is known (Pellicci et al., 2000 J. Immunol.Methods 246 (1-2) p149-163). Briefly, co-expression of CDα with CD8β ledto CD8αβ expression, which was secreted as a non-covalent heterodimer at3 mg/l in the presence of CD8αα. In order to separate the CD8α homodimerfrom the CD8αβ heterodimer, affinity chromatographic techniques specificfor the CD8β subunit were employed. The inclusion of a hexahistidine tagat the C-terminus of CD8β enabled affinity purification of soluble CD8αβ(and sCD8αα) under neutral conditions, yielding recombinant protein withthe correct stoichiometry and full antigenic activity. This productionmethod is expected to be suitable for the production of CD8αβheterodimers of the present invention.

[0060] Modified CD8 molecules of the present invention can be expressedas soluble recombinant protein for extracellular addition, or expressedintracellularly by transfection of a DNA construct encoding the modifiedCD8 molecule. Transfection of DNA can be achieved both in vitro as wellas in vivo, for example by using various type of recombinant viruses asvehicles for DNA transformation or by transfection techniques that use“naked” DNA. For example, an organ to be transplanted may be incubatedin a modified CD8 molecule of the present invention to make it moredifficult for the immune system of host to recognise and reject it.

[0061] The ability of soluble CD8, delivered in addition tochloramphenical acetyl transferase (CAT) via transfection of mice withan adenovirus vector, to inhibit CD8+T cell proliferation and responsesin vivo has been demonstrated. (Peng et al, (2000) Journal of Immunology165:1470-1478). The results of this study demonstrate that transgenicsoluble CD8 continues to be present in the blood of the mice at least 28after injection. The ability of intrahepatic CTLs, target cells takenfrom mice 10 days after injection, to lyse CAT-infected C57SV was alsoassessed. The cells from mice transfected with soluble CD8 resulted inspecific lysis of the target cells a factor of 4 times lower than Tcells from control mice at an E:T ratio of 10:1.

[0062] Preferably in gene therapy, the modified CD8 molecules of thepresent invention are administered such that they are expressed in thesubject to be treated, for example in the form of a recombinant DNAmolecule comprising a polynucleotide encoding the modified CD8 moleculeof the present invention operatively linked to a nucleic acid sequencewhich controls expression, such as in an expression vector. Such avector will thus include appropriate transcriptional control signalsincluding a promoter region capable of expressing the coding sequence,said promoter being operable in the subject to be treated. Thus forhuman gene therapy, the promoter, which term includes not only thesequence necessary to direct RNA polymerase to the transcriptional startsite, but also, if appropriate, other operating or controlling sequencesincluding enhancers, is preferably a human promoter sequence from ahuman gene, or from a gene which is typically expressed in humans, suchas the promoter from human cytomegalovirus (CMV). Among known eukaryoticpromoters suitable in this regard are the CMV immediate early promoter,the HSV thymidine kinase promoter, the early and late SV40 promoters,the promoters of retroviral LTRs, such as those of the Rous sarcomavirus (“RSV”), and metallothionein promoters, such as the mousemetallothionein-I promoter.

[0063] A polynucleotide sequence and transcriptional control sequencemay be provided cloned into a replicable plasmid vector, based oncommercially available plasmids, such as pBR322, or may be constructedfrom available plasmids by routine application of well known, publishedprocedures.

[0064] The vector may also include transcriptional control signals,situated 3′ to the modified CD8 molecule encoding sequence, and alsopolyadenylation signals, recognisable in the subject to be treated, suchas, for example, the corresponding sequences from viruses such as, forhuman treatment, the SV40 virus. Other transcriptional controllingsequences are well known in the art and may be used.

[0065] The expression vectors may also include selectable markers, suchas for antibiotic resistance, which enable the vectors to be propagated.

[0066] Expression vectors capable in situ of synthesising modified CD8molecules of the present invention may be introduced directly byphysical methods. Examples of these include topical application of the“naked” nucleic acid vector in an appropriate vehicle for example insolution in a pharmaceutically acceptable excipient, such as phosphatebuffered saline (PBS). Other physical methods of administering the DNAdirectly to the recipient include ultrasound, electrical stimulation,electroporation and microseeding.

[0067] Nucleic acid sequence encoding modified CD8 molecules of thepresent invention for use in the therapy of the invention may also beadministered by means of delivery vectors. These include viral deliveryvectors, such as adenovirus or retrovirus delivery vectors known in theart. Other non-viral delivery vectors include lipid delivery vectors,including liposome delivery vehicles, known in the art.

[0068] Such a nucleic acid sequence may also be administered by means oftransformed host cells. Such cells include cells harvested from thesubject, into which the nucleic acid sequence is introduced by genetransfer methods known in the art, followed by growth of the transformedcells in culture and administration to the subject.

[0069] Expression constructs such as those described above may be usedin a variety of ways in the therapy of the present invention. Thus, theymay be directly administered to the subject, or they may be used toprepare modified CD8 molecules of the present invention, which can thenbe administered as is discussed in more detail below. The invention alsorelates to host cells which are genetically engineered with constructswhich comprise polynucleotide encoding modified CD8 molecules of thepresent invention, and to the uses of these vectors and cells in thetherapeutic methods of the invention. These constructs may be used perse in the therapeutic methods of the invention or they may be used toprepare a modified CD8 molecule of the present invention for use in thetherapeutic methods of the invention described in greater detail below.

[0070] The vector may be, for example, a plasmid vector, a single ordouble-stranded phage vector, a single or double-stranded RNA or DNAviral vector, depending upon whether the vector is to be administereddirectly (i.e. for in situ synthesis), or is to be used for synthesis ofa modified CD8 molecule. Starting plasmids disclosed herein are eithercommercially available, publicly available, or can be constructed fromavailable plasmids by routine application of well known, publishedprocedures. Many plasmids and other cloning and expression vectors thatcan be used in accordance with the present invention are well known andreadily available to those of skill in the art.

[0071] Generally, vectors for expressing a modified CD8 molecule of thepresent invention for use in the invention comprise cis-acting controlregions effective for expression in a host operatively linked to thepolynucleotide to be expressed. Appropriate trans-acting factors eitherare supplied by the host, supplied by a complementing vector or suppliedby the vector itself upon introduction into the host.

[0072] In certain embodiments in this regard, the vectors provide forspecific expression. For production of modified CD8 molecules of thepresent invention, such specific expression may be inducible expressionor expression only in certain types of cells or both inducible andcell-specific. Particularly preferred among inducible vectors arevectors that can be induced for expression by environmental factors thatare easy to manipulate, such as temperature and nutrient additives. Avariety of vectors suitable to this aspect of the invention, includingconstitutive and inducible expression vectors for use in prokaryotic andeukaryotic hosts, are well known and employed routinely by those ofskill in the art.

[0073] A great variety of expression vectors can be used to expressmodified CD8 molecules for use in the invention. Such vectors include,among others, chromosomal, episomal and virus-derived vectors, e.g.,vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, adeno-associatedviruses, fowl pox viruses, pseudorabies viruses and retroviruses, andvectors derived from combinations thereof, such as those derived fromplasmid and bacteriophage genetic elements, such as cosmids andphagemids, all may be used for expression in accordance with this aspectof the present invention. Generally, any vector suitable to maintain,propagate or express polynucleotides to express a polypeptide in a hostmay be used for expression in this regard.

[0074] The appropriate DNA sequence may be inserted into the vector byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

[0075] The nucleic acid sequence in the expression vector may beoperatively linked to appropriate expression control sequence(s),including, for instance, a promoter to direct mRNA transcription.Representatives of such promoters include, but are not limited to, thephage lambda PL promoter, the E. coli lac, trp and tac promoters, forrecombinant expression, and the SV40 early and late promoters andpromoters of retroviral LTRs for in situ expression.

[0076] In general, expression constructs will contain sites fortranscription initiation and termination, and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will include atranslation initiating AUG at the beginning and a termination codonappropriately positioned at the end of the polypeptide to be translated.

[0077] In addition, the constructs may contain control regions thatregulate as well as engender expression. Generally, in accordance withmany commonly-practised procedures, such regions will operate bycontrolling transcription, such as transcription factors, repressorbinding and termination sites, among others.

[0078] Vectors for propagation and expression generally will includeselectable markers and amplification regions, such as, for example,those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989).

[0079] Representative examples of appropriate hosts for recombinantexpression of CD8 molecules of the present invention include bacterialcells, such as streptococci, staphylococci, E. coli, streptomyces andBacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal or human cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293and Bowes melanoma cells; and plant cells.

[0080] The following vectors, which are commercially available, areprovided by way of example. Among vectors preferred for use in bacteriaare pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors,Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540,pRIT5 available from Pharmacia, and pBR322 (ATCC 37017). Among preferredeukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG availablefrom Stratagene; and pSVK3, pBPV, pMSG and pSVL available fromPharmacia. These vectors which can be used both for recombinantexpression and for in situ expression are listed solely by way ofillustration of the many commercially available and well known vectorsthat are available to those of skill in the art for use in accordancewith this aspect of the present invention. It will be appreciated thatany other plasmid or vector suitable for, for example, introduction,maintenance, propagation or expression of a polynucleotide orpolypeptide for use in the therapy of the invention in a host may beused in this aspect of the invention.

[0081] Examples of vectors for use in this aspect of the inventioninclude expression vectors in which cDNA sequence encoding a modifiedCD8 molecule of the present invention is inserted in a plasmid wherebygene expression is driven from the human immediate early cytomegalovirusenhancer-promoter (Foecking and Hofstetter, Cell, 45, 101-105, 1986).Such expression plasmids may contain SV40 RNA processing signals such aspolyadenylation and termination signals. Expression constructs which usethe CMV promoter and that are commercially available are pCDM8, pcDNA1and derivatives, pcDNA3 and derivatives (Invitrogen). Other expressionvectors available which may be used are pSVK3 and pSVL which contain theSV40 promoter and mRNA splice site and polyadenylation signals from SV40(pSVK3) and SV40 VP1 processing signals (pSVL; vectors from Pharmacia).

[0082] Promoter regions can be selected from any desired gene usingvectors that contain a reporter transcription unit lacking a promoterregion, such as a chloramphenicol acetyl transferase (“CAT”)transcription unit, downstream of restriction site or sites forintroducing a candidate promoter fragment; i.e., a fragment that maycontain a promoter. As is well known, introduction into the vector of apromoter-containing fragment at the restriction site upstream of the catgene engenders production of CAT activity, which can be detected bystandard CAT assays. Vectors suitable to this end are well known andreadily available, such as pKK232-8 and pCM7. Promoters for expressionof polynucleotides for use in the therapy of the present inventioninclude not only well known and readily available promoters, but alsopromoters that readily may be obtained by the foregoing technique, usinga reporter gene; for in situ expression, such a promoter should berecognised in the subject to be treated.

[0083] Among known prokaryotic promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the therapy of thepresent invention are the E. coli lacI and lacZ and promoters, the T3and T7 promoters, the gpt promoter, the lambda PR, PL promoters and thetrp promoter.

[0084] Recombinant expression vectors will include, for example, originsof replication, a promoter preferably derived from a highly-expressedgene to direct transcription of a downstream structural sequence, and aselectable marker to permit isolation of vector containing cells afterexposure to the vector.

[0085] Polynucleotides for use in the therapy of the invention generallywill be inserted into the vector using standard techniques so that it isoperably linked to the promoter for expression. The polynucleotide willbe positioned so that the transcription start site is locatedappropriately 5′ to a ribosome binding site. The ribosome binding sitewill be 5′ to the AUG that initiates translation of the polypeptide tobe expressed. Generally, there will be no other open reading frames thatbegin with an initiation codon, usually AUG, and lie between theribosome binding site and the initiation codon. Also, generally, therewill be a translation stop codon at the end of the polypeptide and therewill be a polyadenylation signal in constructs for use in eukaryotichosts. Transcription termination signal appropriately disposed at the 3′end of the transcribed region may also be included in the polynucleotideconstruct.

[0086] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide when recombinantlysynthesised. These signals may be endogenous to the polypeptide or theymay be heterologous signals.

[0087] The polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals but alsoadditional heterologous functional regions. Thus, for instance, a regionof additional amino acids, particularly charged amino acids, may beadded to the N- or C-terminus of the polypeptide to improve stabilityand persistence in the host cell, during purification or duringsubsequent handling and storage. Also, a region may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability or to facilitate purification, among others, are familiar androutine techniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilise orpurify polypeptides. Cells typically then are harvested bycentrifugation, disrupted by physical or chemical means, and theresulting crude extract retained for further purification.

[0088] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well known to those skilled in the art.

[0089] Mammalian expression vectors may comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation regions, splice donor andacceptor sites, transcriptional termination sequences, and 5′ flankingnon-transcribed sequences that are necessary for expression.

[0090] For preparing a modified CD8 molecule of the present invention,genetically engineered host cells may be used. Introduction of apolynucleotide into the host cell can be affected by calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction, infection or othermethods. Such methods are described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

[0091] Mature proteins can be expressed in host cells includingmammalian cells such as CHO cells, yeast, bacteria, or other cells underthe control of appropriate promoters. Cell-free translation systems canalso be employed to produce such proteins using RNAs derived from theDNA constructs of the present invention. Appropriate cloning andexpression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL,2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989).

[0092] The polypeptide can be recovered and purified from recombinantcell cultures by well-known methods including ammonium sulphate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography is employed for purification. Well known techniques forrefolding protein may be employed to regenerate active conformation whenthe polypeptide is denatured during isolation and or purification.

[0093] Binding of CD8 to MHC/peptide complexes can be convenientlydetected by surface plasmon resonance studies, for instance on theBiacore2000 or Biacore3000 systems (Garcia, et al. Nature 384: 577-81Issn: 0028-0836 (1996); Wyer, et al. Immunity 10: 219-225 (1999)). Theproduction of soluble MHC-peptide complexes is well known. SolubleMHC-peptide complexes were first obtained by cleaving the molecules ofthe surface of antigen presenting cells with papain (Bjorkman, et al. JMol Biol 186: 205-10 (1985)). Although this approach provided materialfor crystallisation, it has, for class I molecules, in recent years beenreplaced by individual expression of heavy and light chain in E. colifollowed by refolding in the presence of synthetic peptide (Gao, et al.Prot. Sci. 7: 1245-49 (1998); Gao, et al. Nature 387: 630-4 (1997);Garboczi, et al. Proc Natl Acad Sci USA 89: 3429-33 Issn: 0027-8424(1992); Garboczi, et al. J Mol Biol 239: 581-7 Issn: 0022-2836 (1994);Madden, et al. [published erratum appears in Cell 1994 Jan. 28;76(2):following 410]. Cell 75: 693-708 Issn: 0092-8674 (1993); Reid, et al. JExp Med 184: 2279-86 (1996); Reid, et al. FEBS Lett 383: 119-23 (1996);Smith, et al. Immunity 4: 215-28 Issn: 1074-7613 (1996); Smith, et al.Immunity 4: 203-13 Issn: 1074-7613 (1996)). This approach has severaladvantages over previous methods in that a better yield is obtained at alower cost, peptide identity can be controlled very accurately, and thefinal product is more homogenous. Furthermore, expression of modifiedheavy or light chain, for instance fused to a protein tag, can be easilyperformed.

[0094] The inhibitory effects of modified CD8 molecules of the presentinvention can also be tested in in vitro CTL assays in order to assesstheir inhibitory effect on T cell activation. These studies can beextended to in vivo analysis of the effects of the modified CD8molecules by testing these in relevant animal disease models.

[0095] The modified CD8 molecules of the invention, and the nucleicacids encoding them, find particular use in the treatment of patientsrequiring immunosuppressive therapy. Such patients include transplantpatients, either awaiting transplant, undergoing transplantation orafter transplantation has taken place. Autoimmune diseases (such asthose described herein) and allergies may also usefully be treated byimmunosuppressive therapy. One specific example is exacerbated asthma inwhich T cells come into play as a result of viral infection. Severedamage to the lungs follows and the result is chronic asthma which canlead to death. The current treatment is with corticosteroids whichstrongly suppress the immune system. A preferable treatment is one whichsuppresses the immune system more selectively, such as specific blockingof CTL function by modified CD8 as described herein.

[0096] The modified CD8 will be administered in a manner appropriate forthe condition to be treated or prevented. For example, for prevention ofgraft rejection one or more injections into the local area concerned maybe most suitable. On the other hand, for an autoimmune disease where theeffects are throughout the body, it may be more appropriate to injectthe modified CD8 directly into the bloodstream.

[0097] Suitable compositions and dosage of modified CD8 as animmunosuppressive agent can be devised by one of ordinary skill in theart. Two or more doses of a smaller amount of modified CD8 may bepreferable to a single high level dose. Formulations may be for exampleliquid formulations, or powder formulations such as those designed fordelivery by a high velocity needle-less delivery device.

[0098] The compositions according to the invention may further comprise,or be administered in a treatment regime with, other agents, inparticular, other immunosuppressive agents. Thus, the invention providesa product containing a modified CD8 molecule as described herein or anucleic acid encoding such a modified CD8 molecule and animmunosuppressive agent as a combined preparation for simultaneous,sequential or separate use in inhibiting CD8⁺ T cell response.

[0099] The most commonly-used immunosuppressive drugs currently includecorticosteroids and more potent inhibitors like, for instance,methotrexate, sulphasalazine, hydroxychloroquine, 6-MP/azathioprine andcyclosporine (Baert & Rutgeerts, 1997, Acta Clin. Belg. 52: 251-7;Singer & McCune, 1998, Curr Opin Rheumatol, 10: 169-73). All of thesetreatments have severe side-effects related to toxicity, however, andthe need for drugs that would allow their elimination from, or reductionin, use is urgent (McKendry, 1997, Rheum Dis ClinNorth Am, 23: 939-54;Ortiz et al., 1998, J. Rheumatol, 25: 36-43; Sibilia et al., 1998, RevRhum Engl Ed 65: 267-73; Singer & McCune, 1998, Curr Opin Rheumatol, 10:169-73). Many immunosuppressive drugs have been found to have greaterefficacy when used in combination, even when the total dose is lowered(Verhoeven et al., 1998, Br J Rheumatol, 37: 612-9).

[0100] Other immunosuppressive drugs include the gentler, but lesspowerful non-steroid treatments such as Aspirin and Ibuprofen, and a newclass of reagents which are based on more specific immune modulatorfunctions. This latter class includes interleukins, cytokines,recombinant adhesion molecules and monoclonal antibodies (for reviewssee Baert & Rutgeerts, 1997, Acta Clin. Belg. 52: 251-7; Chatenoud,1998, Mol Med Today, 4: 25-30).

[0101] CD8αα is specific for class I MHC molecules and is thereforeexpected to inhibit only the response of cytotoxic or memory T cells totarget cells presenting class I complexes. Many cellular immuneresponses are of a composite nature, involving class I and classII-restricted T cells. In some situations, it may be desirable to usemodified CD8αα on its own to suppress unwanted CTL responses. In manysituations, modified CD8αα may be useful in combination with otherimmunosuppressive drugs or reagents which suppress other elements of theimmune response.

[0102] It is envisaged that including modified CD8 in animmunosuppressive treatment protocol will increase the efficiency ofimmunosuppression, and particularly, may enable the administered amountsof other drugs, which have toxic or other adverse effects to bedecreased.

[0103] Medicaments in accordance with the invention will usually besupplied as part of a sterile, pharmaceutical composition which willnormally include a pharmaceutically acceptable carrier. Thispharmaceutical composition may be in any suitable form, (depending uponthe desired method of administering it to a patient).

[0104] It may be provided in unit dosage form, will generally beprovided in a sealed container and may be provided as part of a kit.Such a kit would normally (although not necessarily) includeinstructions for use. It may include a plurality of said unit dosageforms.

[0105] The pharmaceutical composition may be adapted for administrationby any appropriate route, for example by the oral (including buccal orsublingual), rectal, nasal, topical (including buccal, sublingual ortransdermal), vaginal or parenteral (including subcutaneous,intramuscular, intravenous or intradermal) route. Such compositions maybe prepared by any method known in the art of pharmacy, for example byadmixing the active ingredient with the carrier(s) or excipient(s) understerile conditions.

[0106] Pharmaceutical compositions adapted for oral administration maybe presented as discrete units such as capsules or tablets; as powdersor granules; as solutions, syrups or suspensions (in aqueous ornon-aqueous liquids; or as edible foams or whips; or as emulsions)

[0107] Suitable excipients for tablets or hard gelatine capsules includelactose, maize starch or derivatives thereof, stearic acid or saltsthereof. Suitable excipients for use with soft gelatine capsules includefor example vegetable oils, waxes, fats, semi-solid, or liquid polyolsetc.

[0108] For the preparation of solutions and syrups, excipients which maybe used include for example water, polyols and sugars. For thepreparation of suspensions oils (e.g. vegetable oils) may be used toprovide oil-in-water or water in oil suspensions.

[0109] Pharmaceutical compositions adapted for transdermaladministration may be presented as discrete patches intended to remainin intimate contact with the epidermis of the recipient for a prolongedperiod of time. For example, the active ingredient may be delivered fromthe patch by iontophoresis as generally described in PharmaceuticalResearch, 3(6):318 (1986).

[0110] Pharmaceutical compositions adapted for topical administrationmay be formulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils. For infections of theeye or other external tissues, for example mouth and skin, thecompositions are preferably applied as a topical ointment or cream. Whenformulated in an ointment, the active ingredient may be employed witheither a paraffinic or a water-miscible ointment base. Alternatively,the active ingredient may be formulated in a cream with an oil-in-watercream base or a water-in-oil base. Pharmaceutical compositions adaptedfor topical administration to the eye include eye drops wherein theactive ingredient is dissolved or suspended in a suitable carrier,especially an aqueous solvent. Pharmaceutical compositions adapted fortopical administration in the mouth include lozenges, pastilles andmouth washes.

[0111] Pharmaceutical compositions adapted for rectal administration maybe presented as suppositories or enemas.

[0112] Pharmaceutical compositions adapted for nasal administrationwherein the carrier is a solid include a coarse powder having a particlesize for example in the range 20 to 500 microns which is administered inthe manner in which snuff is taken, i.e. by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable compositions wherein the carrier is a liquid, foradministration as a nasal spray or as nasal drops, include aqueous oroil solutions of the active ingredient.

[0113] Pharmaceutical compositions adapted for administration byinhalation include fine particle dusts or mists which may be generatedby means of various types of metered dose pressurised aerosols,nebulizers or insufflators.

[0114] Pharmaceutical compositions adapted for vaginal administrationmay be presented as pessaries, tampons, creams, gels, pastes, foams orspray formulations.

[0115] Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solution which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe formulation substantially isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. Excipients which may beused for injectable solutions include water, alcohols, polyols,glycerine and vegetable oils, for example. The compositions may bepresented in unit-dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carried, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules and tablets.

[0116] The pharmaceutical compositions may contain preserving agents,solubilising agents, stabilising agents, wetting agents, emulsifiers,sweeteners, colourants, odourants, salts (substances of the presentinvention may themselves be provided in the form of a pharmaceuticallyacceptable salt), buffers, coating agents or antioxidants. They may alsocontain therapeutically active agents in addition to the substance ofthe present invention.

[0117] Dosages of the substances of the present invention can varybetween wide limits, depending upon the disease or disorder to betreated, the age and condition of the individual to be treated, etc. anda physician will ultimately determine appropriate dosages to be used.The dosage may be repeated as often as appropriate. If side effectsdevelop the amount and/or frequency of the dosage can be reduced, inaccordance with normal clinical practice.

[0118] In a further aspect, the soluble CD8 mutants described hereinfind application as a screening reagent. The relatively weak affinity ofnative soluble CD8 for HLA molecules places stringent requirements onthe technology required to screen for inhibition of the CD8-HLAinteraction. Many high throughput screen (HTS) assays involve homogenousmethodologies, such as homogeneous time resolved fluorescence (HTRF).Such techniques have drawbacks when approaching low affinityinteractions, as they require relatively high concentrations of theradioactive or fluorescent tracer molecules, typically resulting in lowsignal to background ratios. The relatively high affinity of the CD8mutants described herein for HLA can result in a higher responses andimproved signal to noise ratios. The AlphaScreen™ (Amplified LuminescentProximity Homogenous Assay), recently developed by Packard, is anon-radioactive homogeneous assay technology specifically applicable tolow affinity interactions. This technique provides an attractive optionon which to base HTS assays for the identification of inhibitors ofinteractions between TCR/CD8 and CD8/HLA.

[0119] Preferred features of each aspect of the invention are as foreach of the other aspects mutatis mutandis. The prior art documentsmentioned herein are incorporated to the fullest extent permitted bylaw.

EXAMPLES Example 1 Ser₅₃→Asn Mutation of Human sCD8αα

[0120] This example describes the construction of a DNA expressionplasmid, pEX103, that codes for a CD8α recombinant protein in whichSerine₅₃ is substituted for Asparagine.

[0121] A DNA plasmid, pBJ112 (described in W0 99/21576) encodes aminoacids 1-120 of human CD8α in which the signal peptide is substituted fora single Methionine residue in order to allow initiation of translationwhen expressed in bacteria.

[0122] pEX103 was generated as follows. PCR mutagenesis was performedwith pBJ112 as a template with the following primers in order to producethe Ser₅₃→Asn mutant of soluble CD8α: Amino Acid    Leu Leu Tyr Leu AsnGln Asn Lys Forward: 5′ CTC CTA TAC CTC AA C CAA AAC AAG CC Reverse:5′  GG CTT GTT TTG G TT  GAG GTA TAG GAG

[0123] Bases shown in bold indicate the codons that were changed toproduce the Ser₅₃→Asn substitution. The bases which were changed inthese codons are underlined.

[0124] 25 ng of plasmid pBJ112 was mixed with 5 μl 10 mM dNTP, 25 μl10×Pfu-buffer (Stratagene), 10 units Pfu polymerase (Stratagene) and thefinal volume was adjusted to 240 μl with H₂O. 48 μl of this mix wassupplemented with 125 ng of each primer diluted to give a finalconcentration of 0.2 μM in 50 μl final reaction volume. After an initialdenaturation step of 2 minutes at 95° C. the reaction mixture wassubjected to 18 rounds of denaturation (95° C., 30 sec.), annealing (55°C., 60 sec.), and elongation (68° C., 10 min.) in a Hybaid PCR expressPCR machine. The product was then digested for 90 minutes at 37° C. with10 units of DpnI restriction enzyme (New England Biolabs) in order toremove the methylated pBJ112 template plasmid. 10 μl of the digestedreaction was transformed into XL1-Blue bacteria and grown for 18 hoursat 37° C. on a plate. A single colony was picked and grown over night in5 ml TYP+Ampicillin (16 g/l Bacto-Tryptone, 16 g/l Yeast Extract, 5 g/lNaCl, 2.5 g/l K₂HPO₄, 100 mg/l Ampicillin). Plasmid DNA was purified ona Qiagen mini-prep column according to the manufacturer's instructionsand the sequence was verified by automated sequencing at the sequencingfacility of Department of Biochemistry, Oxford University.

Example 2 Gln₂→Lys Mutation of Human sCD8αα

[0125] This example describes the construction of a DNA expressionplasmid, pEX106, that codes for sCD8αα in which Glutamine₂ issubstituted for Lysine. The DNA sequences of the primers used are shownbelow: Amino Acid:    Asp Ile His Met Ser Lys Phe Arg Val Forward:5′ GAT ATA CAT ATG AGT A AA TTT CGT GTA TC Reverse: 5′  GA TAC ACG AAATT T  ACT CAT ATG TAT ATC

[0126] Mutation of DNA plasmid pBJ112 was carried out according to thesame protocol as described in Example 1, and the sequence was verifiedby automated sequencing at the sequencing facility of Department ofBiochemistry, Oxford University.

Example 3 Asn₂₈→Gln Mutation of Human sCD8αα

[0127] This example describes the construction of the DNA expressionplasmid, pEX107, that codes for sCD8αα in which Asparagine₂₈ issubstituted for Glutamine. The DNA sequences of the primers used areshown below: Amino Acid:      Leu Leu Ser Gln Pro Thr Ser Forward: 5′ GCTG CTG TCC C A G  CCG ACG TCG G Reverse: 5′ C CGA CGT CGG C T G  GGACAG CAG C

[0128] Mutation of DNA plasmid pBJ112 was carried out according to thesame protocol as described in Example 1, and the sequence was verifiedby automated sequencing at the sequencing facility of Department ofBiochemistry, Oxford University.

Example 4 Phe₄₈→Glu Mutation of Human sCD8αα

[0129] This example describes the construction of a DNA expressionplasmid, pEX108, that codes for sCD8αα in which Phenylalanine₄₈ issubstituted for Glutamic acid. The DNA sequences of the primers used areshown below: Amino Acid:       Ser Pro Thr Glu Leu Leu Tyr Forward:5′ CC AGT CCC ACC GAA  CTC CTA TAC C Reverse: 5′  G GTA TAG GAG TTC  GGTGGG ACT GG

[0130] Mutation of DNA plasmid pBJ112 was carried out according to thesame protocol as described in Example 1, and the sequence was verifiedby automated sequencing at the sequencing facility of Department ofBiochemistry, Oxford University.

Example 5 Leu₉₇→Tyr Mutation of Human sCD8αα

[0131] This example describes the construction of a DNA expressionplasmid, pEX109, that codes for sCD8αα in which Leucine₉₇ is substitutedfor Tyrosine. The DNA sequences of the primers used are shown below:Amino Acid:       Cys Ser Ala Tyr Ser Asn Ser Forward: 5′ TC TGC TCG GCCTAT  AGC AAC TCC A Reverse: 5′  T GGA GTT GCT ATA  GGC CGA GCA GA

[0132] Mutation of DNA plasmid pBJ112 was carried out according to thesame protocol as described in Example 1, and the sequence was verifiedby automated sequencing at the sequencing facility of Department ofBiochemistry, Oxford University.

Example 6 Leu₉₇→Gln Mutation of Human sCD8αα

[0133] This example describes the construction of a DNA expressionplasmid, pEX110, that codes for sCD8αα in which Leucine₉₇ is substitutedfor Glutamine. The DNA sequences of the primers used are shown below:Amino Acid:      Cys Ser Ala Gln Ser Asn Ser Forward: 5′ C TGC TCG GCC CA G AGC AAC TCC Reverse: 5′   GGA GTT GCT C T G GGC CGA GCA G

[0134] Mutation of DNA plasmid pBJ112 was carried out according to thesame protocol as described in Example 1, and the sequence was verifiedby automated sequencing at the sequencing facility of Department ofBiochemistry, Oxford University.

Example 7 Leu₉₇→Ser Mutation of Human sCD8αα

[0135] This example describes the construction of a DNA expressionplasmid, pEX111, that codes for sCD8αα in which Leucine₉₇ is substitutedfor Serine. The DNA sequences of the primers used are shown below: AminoAcid:      Cys Ser Ala Ser Ser Asn Ser Forward: 5′ C TGC TCG GCC TCG AGC AAC TCC A Reverse: 5′ T GGA GTT GCT C GA  GGC CGA GCA G

[0136] Mutation of DNA plasmid pBJ112 was carried out according to thesame protocol as described in Example 1, and the sequence was verifiedby automated sequencing at the sequencing facility of Department ofBiochemistry, Oxford University.

Example 8 Asn₉₉→Ile Mutation of Human sCD8αα

[0137] This example describes the construction of a DNA expressionplasmid pEX112 that codes for sCD8αα in which Asparagine₉₉ issubstituted for Isoleucine. The DNA sequences of the primers used areshown below: Amino Acid:       Ala Leu Ser Ile Ser Ile Met Forward:5′ CG GCC CTG AGC A T C TCC ATC ATG T Reverse: 5′  A CAT GAT GGA G AT GCT CAG GGC CG

[0138] Mutation of DNA plasmid pBJ112 was carried out according to thesame protocol as described in Example 1, and the sequence was verifiedby automated sequencing at the sequencing facility of Department ofBiochemistry, Oxford University.

Example 9 Asn₉₉→Met Mutation of Human sCD8αα

[0139] This example describes the construction of a DNA expressionplasmid, pEX113, that codes for sCD8αα in which Asparagine₉₉ issubstituted for Methionine. The DNA sequences of the primers used areshown below: Amino Acid:       Ala Leu Ser Met Ser Ile Met Forward:5′  G GCC CTG AGC A TG  TCC ATC ATG TA Reverse: 5′ TA CAT GAT GGA CAT GCT CAG GGC C

[0140] Mutation of DNA plasmid pBJ112 was carried out according to thesame protocol as described in Example 1, and the sequence was verifiedby automated sequencing at the sequencing facility of Department ofBiochemistry, Oxford University.

Example 10 Cys₃₃→Ala Mutation of Human sCD8αα

[0141] This example describes the construction of the DNA expressionplasmid pEX115 that codes for the sCD8αα in which Cysteine₃₃ issubstituted for Alanine. The DNA sequences of the primers used are shownbelow (nucleotide substitutions are indicated in bold): Amino Acid:     Thr Ser Gly Ala Ser Trp Leu Forward: 5′-G ACG TCG GGC GC C TCG TGGCTC-3′ Reverse: 5′-GAG CCA CGA G GC  GCC CGA CGT C-3′

[0142] Mutation of DNA plasmid pBJ112 was carried out according to thesame protocol as described in Example 1 and the sequence was verified byautomated sequencing at the sequencing facility of Department ofBiochemistry, Oxford University.

Example 11 Cys₃₃→Ser Mutation of Human sCD8αα

[0143] This example describes the construction of the DNA expressionplasmid pJMB017 that codes for the sCD8αα in which Cysteine₃₃ issubstituted for Serine. The DNA sequences of the primers used are shownbelow (nucleotide substitutions are indicated in bold): Amino Acid:     Thr Ser Gly Ser Ser Trp Leu Forward: 5′-G ACG TCG GGC A GC TCG TGGCTC-3′ Reverse: 5′-GAG CCA CGA GC T  GCC CGA CGT C-3′

[0144] Mutation of DNA plasmid pBJ112 was carried out according to thesame protocol as described in Example 1 and the sequence was verified byautomated sequencing at the sequencing facility of Department ofBiochemistry, Oxford University.

Example 12 Expression, Refolding and Purification of Human sCD8ααMutants

[0145] sCD8α protein was expressed from the DNA vector pBJ112 and frommutated derivatives of pBJ112, i.e. pEX013, pEX106-113, PEX115 andpJMB107, in the E. coli strain BL21-DE3 pLysS (Novagen). pBJ112 containsthe sCD8α gene under the control of the strongly inducible T7 promoterin the vector pGMT7 (Studier, et al. Methods in Enzymology 185: 60-89ISSN: 0076-6879 (1990)). BL21 cells transformed with the sCDα expressingvectors were plated on LB/agar/100 mg/l Ampicillin plates made accordingto a standard recipe. Transformants were then grown in TYP medium withAmpicillin (16 g/l Bacto-Tryptone, 16 μl Yeast Extract, 5 g/l NaCl, 2.5g/l K₂HPO₄, 100 mg/l Ampicillin) to an OD₆₀₀˜0.5 (RANGE 0.4-0.6). Forlarge-scale expression, 1 l volumes of TYP media were prepared in 2 lconical flasks and were covered with four layers of aluminium foil andwere autoclaved. Cell densities were measured using optical density at600 nm wavelength (OD600) on a Beckman DU530 spectrophotometer. SterileTYP media was used as a blank. Inclusion bodies were purified asdescribed (Gao, et al, Prot. Sci.7: 1245-49 (1998)). Cells were lysed byincubation for 30 minutes at room temperature in ‘Lysis Buffer’ (10 mMEDTA (from 0.5 M stock pH 8.0), 2 mM DTT (from 1 M stock in 10 mM sodiumacetate pH 5.2, stored at −20° C.), 10 mM Tris pH 8.1 (from 2 M stock pH8.1), 150 mM NaCl (from 4 M stock), 200 μg/ml lysozyme (from 20 mg/mlstock stored at −20° C.), 10% glycerol (from fluid), 2500 units ofDNAase 1 and 10 mM MgCl₂ using a 50 ml Dounce homogeniser DNase I andlysozyme were from Sigma). Sonication, in lysis buffer, to break openthe cells was performed using a 12 mM probe sonicator (Milsonix XL2020).The probe was tuned according to the manufacturers instructions. Theresulting suspension was then diluted 1:1 in ‘Triton Buffer’ (0.5% (w/v)Triton X-100 (from fluid), 50 mM Tris pH 8.1 (from 2 M stock), 100 mMNaCl (from 5 M stock), 0.1% sodium azide (from solid), 10 mM EDTA (from0.5 M stock pH 8.0), 2 mM DTT (from 1 M stock in 10 mM sodium acetate pH5.2, stored at −20° C.), and left overnight. The inclusion bodies wereseparated from cell debris by centrifugation in a Beckman J2-21centrifuge equipped with a JA-20 rotor as described (Gao, et al, Prot.Sci.7: 1245-49 (1998)) and stored at −20° C. Inclusion bodies were thenthawed and resuspended in ‘Resuspension Buffer’ (50 mM Tris pH 8.1 (from2 M stock), 100 mM NaCl (from 4 M stock), 10 mM EDTA (from 0.5 M stockpH 8.0), 2 mM DTT (from 1 M stock in 10 mM sodium acetate pH 5.2, storedat −20° C.)), and denatured in 6M Guanidine and 10 mM DTT buffered withTris-HCl pH 8.1 (all chemicals from Sigma).

[0146] The sCD8αα proteins were then refolded in vitro in the presenceof 0.4M L-Arginine and purified by ion-exchange (PDKOS HS column) and/orgel filtration chromatography, for instance on a Pharmacia Superdex 75column. The 99N→I and 99N→M sCD8αα mutants failed to refold correctlyand therefore were not further assessed.

Example 13 Mammalian Expression Vectors Coding for Soluble CD8α

[0147] A. Native CD8α Lacking the Transmembrane Domain (Natural SpliceVariant) Primers: Signal peptide forward primer 5′-CCCCTCTAGA TGGCCTTACCAGTGACCGCC-3′ Cytoplasmic tail reverse primer 5′-GGGGAATTCT TAGACGTATCTCGCCGAAAG GCT-3′

[0148] Using lymphocyte cDNA as template, PCRs is set up with primers at0.5 μM each, dNTP at 0.2 mM each, and Pfu DNA polymerase at 0.05 U/μl in1×Pfu buffer as provided from the supplier of the polymerase and run asfollows: 10 minutes initial denaturation (94 C), followed by 20 cyclesof denaturation (1 minute, 94 C); annealing (1 min, 55 C); elongation (3minutes, 73 C); and a final elongation step for 10 minutes at 73 C.

[0149] Two dominant species are amplified, a full length product and ashorter product derived from a splice variant. This variant accounts forapproximately 15% of the total CD8α mRNA in human CTLs. (Norment et al,.1989, J Immunol, 142(9): 3312-9).

[0150] The short 615 base pair PCR fragment is purified and subclonedinto the mammalian expression vector pcDNA3.1-(™ Invitrogen) between theXbaI and EcoRI restriction sites by standard techniques to give pEX119.In this plasmid, mammalian expression is controlled by a strongconstitutive Cytomegalovirus (CMV) enhancer-promoter.

[0151] B. CD8-α_(S53) 1-120. Primers: Signal peptide forward primer5′-CCCCTCTAGA TGGCCTTACC AGTGACCGCC-3′ aa. 120 reverse primer5′-GGGGAATTCT ATGGCGTCGT GGTGGG-3′

[0152] Using lymphocyte cDNA as template, PCR is set up with primers at0.5 μM each, dNTP at 0.2 mM each, and Pfu DNA polymerase at 0.05 U/μl in1×Pfu buffer as provided from the supplier of the polymerase and run asfollows: 10 minutes initial denaturation (94 C), followed by 20 cyclesof denaturation (1 minute, 94 C); annealing (1 min, 55 C); elongation (3minutes, 73 C); a final elongation step for 10 minutes at 73 C.

[0153] The resulting 443 base pair PCR fragment is purified andsubcloned into pcDNA3.1-(™ Invitrogen) between the XbaI and EcoRIrestriction sites by standard techniques to give pEX120.

[0154] C. CD8-α_(N53) w/o Transmembrane Domain (Splice Variant)

[0155] pEX501 is made by PCR mutagenesis (in the same manner describedin Example 1) using the primers shown and pEX119 as template. Thisplasmid codes for the natural CD8-α splice variant with a single pointmutation Ser₅₃ Asn. Amino Acid    Leu Leu Tyr Leu Asn Gln Asn LysForward: 5′ CTC CTA TAC CTC AA C CAA AAC AAG CC Reverse: 5′  GG CTT G TT TTG GTT GAG GTA TAG GAG

[0156] D. CD8-α_(N53) 1-120.

[0157] pEX502 is made by PCR mutagenesis (in the same manner describedin Example 1) using the primers shown on pEX120. This plasmid codes forCD8-α amino acids −21 to 120 with a single point mutation Ser₅₃ Asn.Amino Acid    Leu Leu Tyr Leu Asn Gln Asn Lys Forward: 5′ CTC CTA TACCTC AA C CAA AAC AAG CC Reverse: 5′  GG CTT GTT TTG G TT  GAG GTA TAGGAG

Example 14 Testing of WT and Ser₅₃→Asn Mutant sCD8αα Protein Binding toTax and Flu HLA-A2/β2m Complexes

[0158] sCD8αα Mutant proteins were prepared according to Example 12.

[0159] The refolding of the Tax and Flu HLA-A2/β2m complexes was carriedout as described in (Gao, et al, Prot. Sci.7: 1245-49 (1998)) containinga tag sequence that can be enzymatically biotinylated (Schatz,Biotechnology N Y 11: 1138-43 (1993); Altman, et al Science 274: 94-6(1996); Wyer, et al. Immunity 10: 219-225 (1999)). The complexes werethen biotinylated using the enzyme BirA (O'Callaghan, et al. AnalBiochem 266(1): 9-15 (1999)) to produce Tax and Flu HLA-A2/β2m complexeswhich were biotinylated towards the C-terminus of the HLA-A2 heavychain. These protein complexes were immobilised on astreptavidin-modified BIAcore chip sensor cell in a BIAcore 3000machine. The CD8 proteins were passed through the sensor cell atconcentrations in the range of 0.025-11.5 mg/ml. The binding of WT andmutant sCD8αα to the Tax and Flu HLA-A2/β2m complexes was monitored bysurface plasmon resonance (SPR).

[0160] Determination of the effects on HLA binding of mutationsintroduced in the sCD8αα protein was accomplished by passing sCD8ααprotein through the BIAcore sensor cells and the levels of binding inthese were compared. Any absent, or significant reduction, of mutantsCD8αα binding to peptide-HLA-A2/β2m complex observed, compared to thatobserved for WT sCD8aa, was concluded to demonstrate that the mutationintroduced into the sCDαα protein had affected the ability of theprotein to bind the HLA/β2m complex.

[0161] Both WT and mutant proteins were purified by gel filtration(Superdex 75HR 10/30) immediately prior to the binding experiments.Fractions containing protein pooled together and concentrated using a 10kd cut-off Centriprep Kit (Millipore). The sCD8αα concentrations weremeasured using optical density at 280 nm wavelength (OD280) on a BeckmanDU530 spectrophotometer. (measured in a capillary). sCD8aa WT: Abs280 =13.8 (11.4 mg/ml) sCD8aa 53S → N mutant: Abs280 = 14 (11.6 mg/ml)

[0162] Chip utilised: CM-5 sensor chip.

[0163] Complexes Immobilised on the Chip:

[0164] flowcell 1: Tax-HLA-A2

[0165] flowcell 2: Tax-HLA-A2

[0166] flowcell 3: Flu-HLA-A2

[0167] flowcell 4: blank (no protein bound)

[0168] 10 solutions of different CD8 (WT and mutant) concentrations wereprepared and sent to all the flowcells.

[0169] Responses were recorded and plotted against CD8 concentration andthe points were fitted in the following equation:

Response=P1*[CD8]/(P2+[CD8])

[0170] Where P1 is the calculated response that would occur when allHLA/β2m complexes were bound to CD8 and P2 is the Kd in μM.

[0171] The results are shown in FIGS. 2 and 3. The affinity of sCD8ααmutant Ser₅₃→Asn for the HLA-A2/β2m complexes (both Flu and Tax) isbetween 3 and 4 times higher than that of WT sCD8αα.

Example 15 Testing of Other Mutant sCD8αα Proteins Binding to TaxHLA-A2/β2m Complexes

[0172] The following sCD8αα mutants were expressed and refolded asdescribed in Example 12. Mutant Plasmid Gln₂ → Lys pEX 106 Asn₂₈ → GlnpEX 107 Phe₄₈ → Glu pEX 108 Leu₉₇ → Tyr pEX 109 Leu₉₇ → Gln pEX 110Leu₉₇ → Ser pEX 111

[0173] The ability of these mutants to bind Tax HLA-A2/β2m complexes wastested as described in Example 14.

[0174] The results obtained are as follows (see also FIG. 4): Mutant Kd(μM) Gln₂ → Lys 363 Asn₂₈ → Gln no binding* Phe₄₈ → Glu no binding*Leu₉₇ → Tyr 630 Leu₉₇ → Gln no binding* Leu₉₇ → Ser no binding*

[0175] None of the mutants assessed in this example produced a sCD8ααmolecule with high binding affinity for Tax HLA-A2.

Example 16 Method for Assessing the Ability of Mutant Soluble CD8αα toInhibit T Cell Activation

[0176] Target cells are grown in RPMI culture medium containing 10%human serum for 5 days. These cells are incubated in RMPI mediumcontaining 1 μM peptide for 2 hours. The target cells are placed intomicrotitre plates with CTL (cytotoxic T lymphocytes) at a range ofEffector: Target cell (E:T) ratios. Supernatants are harvested after2-16 hours.

[0177] Example combinations of Class I HLA molecules and theirrespective T cells: HLA T cell Clone Reference A*0201 AL1.1 Salter etal, 1990 B*08 IM6/LC13 Argaet et al, 1994

[0178] Experimental Design

[0179] Negative Control—Antigen presenting target cells

[0180] Positive Control—Antigen presenting target cells incubated in thepresence of a range (0-100 μg/ml soluble WT CD8αα.

[0181] Test Samples—Antigen presenting target cells incubated in thepresence of a range (0-100 μg/ml soluble mutant CD8αα.

[0182] Assay components for these experiments are:

[0183] 18 μl 10× peptide (10⁻⁵ M)

[0184] 18 μl PBS (−ve control), or PBS with WT soluble CD8αα (+vecontrol), or PBS with mutant soluble CD8αα (test samples)

[0185] 50 μl APC Target Cells (5,000 cells)

[0186] 100 μl containing 5,000-50,000 CTL.

[0187] A standard cytokine assay, for example a macrophage inflammatoryprotein-1β (MIP-1β) assay (Quantikine®—Human M1β Immunoassay, Cat No:DMB00, R&D Systems Europe, Abingdon UK) is the carried out on thesupernatant in accordance with the manufacturers instructions.

[0188] Alternative assays based on the cytokines IFN-γ and RANTES couldalso be used.

[0189] Chemokines are cell activation markers expressed by a range ofcells including CTL. Therefore, any decrease in cytokine productionobserved from the Test samples compared the Controls indicates areduction in T cell activation.

Example 17 Method for Assessing the Ability of Mutant Soluble CD8 toInhibit T Cell Activation

[0190] Target cells are grown in RPMI culture medium containing 10%human serum for 5 days. These cells are incubated in RMPI mediumcontaining 1 μM peptide for 2 hours. The target cells are placed intomicrotitre plates with CTL (cyto-toxic lymphocytes) at a range ofEffector:Target cell (E:T) ratios. Supernatants are harvested after 2-16hours.

[0191] Example combinations of Class I HLA molecules and theirrespective T cells: HLA T cell Clone Reference A*0201 AL1.1 Salter etal, 1990 B*08 IM6/LC13 Argaet et al, 1994

[0192] Experimental Design

[0193] Control—Non-transformed antigen presenting target cells

[0194] Test Samples—Antigen presenting target cells transformed toexpress mutant soluble CD8.

[0195] A standard cytokine assay, for example a macrophage inflammatoryprotein-1β (MIP-1β) assay (Quantikine® Human MIP-1≢ Immunoassay, Cat No:DMB00, R&D Systems Europe, Abingdon UK) is the carried out on thesupernatant in accordance with the manufacturers instructions.

[0196] Alternative assays based on the cytokines IFN-γ and RANTES couldalso be used.

[0197] Chemokines are cell activation markers expressed by a range ofcells including CTL. Therefore, any decrease in cytokine productionobserved from the Test samples compared the controls indicates areduction in T cell activation.

Example 18 Test for Immunogenicity of Mutant β-2-microglobulin

[0198] Mutant soluble CD8αα molecules could potentially induce an immuneresponse either by antibodies and/or T cells. The introduction ofmutation(s) in the CD8αα protein could potentially introduce aconformational change in the protein structure, which would berecognised by antibodies as a structural foreign antigen or,alternatively, enzymatic degradation of mutant soluble CD8αα couldproduce peptides not normally presented by self MHC molecules. Thesemutant peptides would then be recognised as foreign and induce acellular immune response. Therefore, mutants are tested in a transgenicrat model expressing human MHC class I molecule (HLA-B27 heavychain+β-2-microglobulin) as follows.

[0199] 1. Inject transgenic rats with 3-4 mg mutant soluble CD8αα.

[0200] 2. Collect serum from rats after 21 days.

[0201] 3. Analyse serum for the production of anti-mutant soluble CD8ααantibodies by an ELISA.

[0202] The procedure for this ELISA is: a. Bind mutant soluble CD8αα tobottom of well; b. Add serum from transgenic rat, which has been treatedwith mutant soluble CD8αα; c. wash three times with 200 μL of washbuffer; d. add the appropriate concentration of conjugated anti-ratantibody; e. wash three times with 200 μL of wash buffer; f. add 100 μLdetection reagent (Alkaline Phosphatase, substrate pNPP) and readabsorbance at 405 nm. Absorbance readings above negative controlreadings will indicate, that anti-mutant soluble CD8αα antibodies arepresent in the serum.

1. A modified CD8 molecule whose binding to MHC is enhanced compared towild type CD8, wherein Ser₅₃ of at least one CD8α chain thereof ismutated to another amino acid.
 2. A molecule as claimed in claim 1,wherein said molecule is an αα or αβ dimer.
 3. A molecule as claimed inclaim 1 or claim 2, wherein Ser₅₃ is mutated to Asn, Gln, His or Lys. 4.A molecule as claimed in claim 1, 2 or 3, which is derived from humanCD8.
 5. A molecule as claimed in any preceding claim, which is a solubleform of CD8.
 6. A molecule as claimed in claim 5, comprising all or asubstantial part of the CD8 immunoglobulin domain, optionally with allor part of the membrane proximal stalk region.
 7. A molecule as claimedin claim 5 or claim 6, wherein Cys₃₃ of at least one CD8α chain thereofis mutated to another amino acid, preferably Ala or Ser.
 8. A moleculeas claimed in claim 5, 6 or 7, comprising residues 1-120 of the humanCD8α chain.
 9. A molecule as claimed in claim 8, comprising the aminoacid sequence of FIG. 1b.
 10. A multimer of a molecule as claimed in anypreceding claim. 11 A nucleic acid, particularly a DNA, comprising asequence which: (i) encodes a molecule as defined in any one of claims 1to 10; (ii) is an RNA equivalent of the DNA of (i); (iii) iscomplementary to the sequences of (i) or (ii); or (iv) has substantialidentity with the sequences of (i), (ii) or (iii).
 12. A nucleic acid asclaimed in claim 11, comprising the DNA sequence set out in FIG. 1b or 1c (with or without the signal sequence) herein.
 13. A vector comprisinga nucleic acid molecule as claimed in claim 11 or claim
 12. 14. A hostcell including the vector as claimed in claim
 13. 15. A method ofproducing a modified CD8 molecule, which method comprises the steps of:i) effecting expression of a nucleic acid molecule as defined in claim10 or claim 11 in a bacterium or eukaryotic cell and recovering theexpressed protein from a cell culture; and ii) treating the expressedprotein to facilitate its purification and carrying out saidpurification.
 16. A composition comprising a modified CD8 molecule asdefined in any one of claims 1 to 10, or a nucleic acid as defined inclaim 11 or claim 12, together with a pharmaceutically acceptablediluent, excipient or carrier.
 17. A modified CD8 molecule as defined inany one of claims 1 to 10, or a nucleic acid as defined in claim 11 orclaim 12, for use in medicine.
 18. The use of a modified CD8 molecule asdefined in any one of claims 1 to 10, or of a nucleic acid as defined inclaim 11 or claim 12, in the manufacture of a medicament for modulatingCD8⁺ T cell response.
 19. A method of modulating the activation of aCD8⁺ T cell by a class I Major Histocompatibility Complex (MHC), themethod comprising exposing the class I MHC to a modified CD8 molecule asdefined in any one of claims 1 to
 10. 20. A method for the treatment ofan autoimmune disorder hypersensitivity (e.g. allergic reaction),graft-versus-host-disease or graft rejection, comprising administeringto a patient a modified CD8 molecule as defined in any one of claims 1to 10, or a nucleic acid as defined in claim 11 or claim
 12. 21. Aproduct containing a modified CD8 molecule as defined in any one ofclaims 1 to 10 or a nucleic acid as defined in claim 11 or claim 12 andan immunosuppressive agent as a combined preparation for simultaneous,sequential or separate use in modulating CD8⁺ T cell response.